TRPV1 mediates astrocyte activation and interleukin-1ß release induced by hypoxic ischemia (HI).

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1ß expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family. METHODS: Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1ß, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually. RESULTS: Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1ß, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1ß by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1ß release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice. CONCLUSIONS: TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1ß mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h).

Vitexin reduces epilepsy after hypoxic ischemia in the neonatal brain via inhibition of NKCC1.

BACKGROUND: Neonatal hypoxic-ischemic brain damage, characterized by tissue loss and neurologic dysfunction, is a leading cause of mortality and a devastating disease of the central nervous system. We have previously shown that vitexin has been attributed various medicinal properties and has been demonstrated to have neuroprotective roles in neonatal brain injury models. In the present study, we continued to reinforce and validate the basic understanding of vitexin (45 mg/kg) as a potential treatment for epilepsy and explored its possible underlying mechanisms. METHODS: P7 Sprague-Dawley (SD) rats that underwent right common carotid artery ligation and rat brain microvascular endothelial cells (RBMECs) were used for the assessment of Na+-K+-Cl- co-transporter1 (NKCC1) expression, BBB permeability, cytokine expression, and neutrophil infiltration by western blot, q-PCR, flow cytometry (FCM), and immunofluorescence respectively. Furthermore, brain electrical activity in freely moving rats was recorded by electroencephalography (EEG). RESULTS: Our data showed that NKCC1 expression was attenuated in vitexin-treated rats compared to the expression in the HI group in vivo. Oxygen glucose deprivation/reoxygenation (OGD) was performed on RBMECs to explore the role of NKCC1 and F-actin in cytoskeleton formation with confocal microscopy, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide, and FCM. Concomitantly, treatment with vitexin effectively alleviated OGD-induced NKCC1 expression, which downregulated F-actin expression in RBMECs. In addition, vitexin significantly ameliorated BBB leakage and rescued the expression of tight junction-related protein ZO-1. Furthermore, inflammatory cytokine and neutrophil infiltration were concurrently and progressively downregulated with decreasing BBB permeability in rats. Vitexin also significantly suppressed brain electrical activity in neonatal rats. CONCLUSIONS: Taken together, these results confirmed that vitexin effectively alleviates epilepsy susceptibility through inhibition of inflammation along with improved BBB integrity. Our study provides a strong rationale for the further development of vitexin as a promising therapeutic candidate treatment for epilepsy in the immature brain.

Vitexin reduces hypoxia-ischemia neonatal brain injury by the inhibition of HIF-1alpha in a rat pup model.

Previous studies have demonstrated that the early suppression of HIF-1α after hypoxia-ischemia (HI) injury provides neuroprotection. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside), an HIF-1α inhibitor, is a c-glycosylated flavone that has been identified in medicinal plants. Therefore, we hypothesized that treatment with vitexin would protect against HI brain injury. Newborn rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O2 at 37 °C). Vitexin (30, 45 or 60 mg/kg) was administered intraperitoneally at 5 min or 3 h after HI. Vitexin, administered 5 min after HI, was neuroprotective as seen by decreased infarct volume evaluated at 48 h post-HI. This neuroprotection was removed when vitexin was administered 3 h after HI. Neuronal cell death, blood-brain barrier (BBB) integrity, brain edema, HIF-1α and VEGF protein levels were evaluated using a combination of Nissl staining, IgG staining, brain water content, immunohistochemistry and Western blot at 24 and 48 h after HI. The long-term effects of vitexin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests. Vitexin (45 mg/kg) ameliorated brain edema, BBB disruption and neuronal cell death; Upregulation of HIF-1α by dimethyloxalylglycine (DMOG) increased the BBB permeability and brain edema compared to HI alone. Vitexin attenuated the increase in HIF-1α and VEGF. Vitexin also had long-term effects of protecting against the loss of ipsilateral brain and improveing neurobehavioral outcomes. In conclusion, our data indicate early HIF-1α inhibition with vitexin provides both acute and long-term neuroprotection in the developing brain after neonatal HI injury.

TRPV1 promotes repetitive febrile seizures by pro-inflammatory cytokines in immature brain.

Febrile seizure (FS) is the most common seizure disorder in children, and children with FS are regarded as a high risk for the eventual development of epilepsy. Brain inflammation may be implicated in the mechanism of FS. Transient receptor potential vanilloid 1 (TRPV1) is believed to act as a monitor and regulator of body temperature. The role of inflammation in synaptic plasticity mediation indicates that TRPV1 is relevant to several nervous system diseases, such as epilepsy. Here, we report a critical role for TRPV1 in a febrile seizure mouse model and reveal increased levels of pro-inflammatory factors in the immature brain. Animals were subjected to hyperthermia for 30 min, which generates seizures lasting approximately 20 min, and then were used for experiments. To invoke frequently repetitive febrile seizures, mice are exposed to hyperthermia for three times daily at an interval of 4h between every time induced seizure, and a total of 4 days to induce. Behavioral testing for febrile seizures revealed that a TRPV1 knock-out mouse model demonstrated a prolonged onset latency and a shortened duration and seizure grade of febrile seizure when compared with wild type (WT) mice. The expression levels of both TRPV1 mRNA and protein increased after a hyperthermia-induced febrile seizure in WT mice. Notably, TRPV1 activation resulted in a significant elevation in the expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and HMGB1) in the hippocampus and cortex. These data indicate that the reduction of TRPV1 expression parallels a decreased susceptibility to febrile seizures. Thus, preventative strategies might be developed for use during febrile seizures.

Regulation of Kv4.2 A-Type Potassium Channels in HEK-293 Cells by Hypoxia.

We previously observed that A-type potassium currents were decreased and membrane excitability increased in hippocampal dentate granule cells after neonatal global hypoxia associated with seizures. Here, we studied the effects of hypoxia on the function and expression of Kv4.2 and Kv4.3 α subunit channels, which encode rapidly inactivating A-type K currents, in transfected HEK-293 cells to determine if hypoxia alone could regulate IA in vitro. Global hypoxia in neonatal rat pups resulted in early decreased hippocampal expression of Kv4.2 mRNA and protein with 6 or 12 h post-hypoxia. Whole-cell voltage-clamp recordings revealed that similar times after hypoxia (1%) in vitro decreased peak currents mediated by recombinant Kv4.2 but not Kv4.3 channels. Hypoxia had no significant effect on the voltage-dependencies of activation and inactivation of Kv4.2 channels, but increased the time constant of activation. The same result was observed when Kv4.2 and Kv4.3 channels were co-expressed in a 1:1 ratio. These data suggested that hypoxia directly modulates A-type potassium channels of the subfamily typically expressed in principal hippocampal neurons, and does so in a manner to decrease function. Given the role of IA to slow action potential firing, these data are consistent with a direct effect of hypoxia to decrease IA as a mechanism of increased neuronal excitability and promotion of seizures.

Expression of TRPV1 in the C57BL/6 mice brain hippocampus and cortex during development.

Transient receptor potential vanilloid 1 (TRPV1) channel has been found to be expressed in a variety of tissues over the last few years, including the central nervous system (CNS). However, the distribution of TRPV1 in the CNS remains a controversial question. Here, we reveal that the expression of TRPV1 can be detected in the C57BL/6 mouse hippocampus and cortex using real-time PCR and western blot. Beyond that, mRNA and protein expression levels of TRPV1 show dynamic changes during brain development. Compared with the earliest timepoint examined at 2 weeks, the expression levels of mRNA progressively increased at 4 and 8 weeks, peaking at the later timepoint, then declined at 16 weeks but remained elevated. However, compared with 2-week-old mice, the expression levels of the other three groups (4-, 8-, and 16-week-old mice) increased overall. These results indicate that TRPV1 channel expression is detectable in the CNS and it varies during postnatal development.